METHOD FOR PREPARING RECOMBINANT PROTEINS THROUGH REDUCTION OF rnpA GENE EXPRESSION

ABSTRACT

A method is described for improving the production of a difficult-to-express recombinant protein in a recombinant microorganism, and more particularly a method for improving the production of a difficult-to-express recombinant protein by use of a recombinant microorganism into which a gene encoding a target protein and an sRNA against a gene encoding ribonuclease P are introduced. By the disclosed method, expressions of a large recombinant protein, a difficult-to-express protein and a useful protein can be dramatically increased by reducing expression of the rnpA gene.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional under 35 U.S.C. § 120 of U.S. patentapplication Ser. No. 15/533,273 filed Jun. 5, 2017, which in turn is aU.S. national phase under the provisions of 35 U.S.C. § 371 ofInternational Patent Application No. PCT/KR2015/013419 filed Dec. 9,2015, which in turn claims priority of Korean Patent Application No.10-2014-0175780 filed Dec. 9, 2014 and Korean Patent Application No.10-2015-0174828 filed Dec. 9, 2015. The disclosures of U.S. patentapplication Ser. No. 15/533,273, International Patent Application No.PCT/KR2015/013419, Korean Patent Application No. 10-2014-0175780, andKorean Patent Application No. 10-2015-0174828 are hereby incorporatedherein by reference in their respective entireties, for all purposes.

TECHNICAL FIELD

The present invention relates to a method for improving the productionof a recombinant protein in a recombinant microorganism, and moreparticularly to a method for improving the production of a recombinantprotein by use of a recombinant microorganism into which a gene encodinga target protein and an sRNA against a gene encoding ribonuclease P areintroduced.

BACKGROUND ART

The development of genetic manipulation technology has led to manystudies focused on producing large amounts of useful proteins usingbacterial and a variety of animals and plants. Host cells for producinglarge amounts of useful proteins are present, including bacteria such asE. coli, and yeasts such as P. pastoris. Of these host cells, E. colihas been most widely used, and studies thereon have been most frequentlyconducted (Choi et al., Chem. Eng. Sci., 66: 876, 2006; Lee, TrendsBiotechnol., 14:98, 1996).

However, if the useful protein to be produced is larger in size than anaturally occurring protein or is difficult to express, many problemsmay arise. If the size of a protein is large, translation of the proteinmay be difficult due to lack of messanger RNA (mRNA), and thusexpression of the desired full-length protein may be difficult. Inaddition, a recombinant protein that is not naturally present in E. colimay be difficult to express, due to proteolysis and RNase-induceddegradation of mRNA (GoBringer et al., J Bacteriol., 188: 6816, 2006;Olson et al., PLoS Pathog., 7(2): e1001287, 2011; Jung et al., BiochemBiophys Res Commun., 186(3):1463, 1992; Altman et al., Phil Trans RSoc., 366, 2011; Turrini et al., PLos One., 7(3): e32456, 2012).

Accordingly, the present inventors have made extensive efforts todevelop a protein expression system for increasing the production of adifficult-to-express foreign protein, and as a result, have found that,when expression of the rnpA gene (which is a component of ribonucleaseP) in a process of expressing the foreign protein by introducing a geneencoding the foreign protein is reduced, expression of thedifficult-to-express foreign protein increases, thereby completing thepresent invention.

DISCLOSURE OF INVENTION Technical Problem

It is a main object of the present invention to provide a recombinantmicroorganism into which a gene encoding a target protein and an sRNAagainst a gene encoding Ribonuclease P are introduced to increase theproduction of a difficult-to-express foreign protein.

Another object of the present invention is to provide a method forproducing a target protein by culturing the above-described recombinantmicroorganism.

Technical Solution

To achieve the above objects, the present invention provides arecombinant vector for expressing a target protein, which comprises agene encoding the target protein and an sRNA against a gene encodingribonuclease P, and a recombinant microorganism into which therecombinant vector is introduced.

The present invention also provides a recombinant microorganism intowhich a gene encoding a target protein and an sRNA against a geneencoding ribonuclease P are introduced.

The present invention also provides a method for producing a targetprotein, the method comprising the steps of: (a) producing the targetprotein by culturing the above-described recombinant microorganism andinducing expression of the target protein in the recombinantmicroorganism; and (b) recovering the produced target protein.

The present invention also provides a method for producing a targetprotein, comprising the steps of: expressing and producing the targetprotein by culturing a recombinant microorganism into which a geneencoding the target protein is introduced; and recovering the producedtarget protein, wherein expression of ribonuclease P is reduced toincrease expression of the target protein.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 is a gene map of the plasmid pTetly2glyAN-rnpA(sRNA).

FIG. 2 shows the results of analyzing expression levels of a silkprotein consisting of 16 repeats and a silk protein consisting of 32repeats by SDS-PAGE. (lane 1: a marker showing protein standardmolecular weight; lanes 2 and 3: the results of inducing proteinexpression in a stain transformed with the plasmid pSH16 at an OD600 of0.4; lanes 4 and 5: the results of inducing protein expression in astrain transformed with the plasmid pSH16 and pACYC184-rnpA(sRNA) at anOD600 of 0.4; lanes 6 and 7: the results of inducing protein expressionin a strain transformed with the plasmid pSH32 at an OD₆₀₀ of 0.4; andlanes 8 and 9: the results of inducing protein expression in a straintransformed with the plasmid pSH32 and pACYC184-rnpA(sRNA) at an OD600of 0.4.)

FIG. 3 shows the results of analyzing expression levels of a silkprotein consisting of 64 repeats by SDS-PAGE. (lane 1: a marker showingprotein standard molecular weight; lane 2: the result of inducingprotein expression in a strain transformed with pSH64 at an OD600 of0.4; and lane 3: the result of inducing protein expression in a straintransformed with the plasmid pSH64 and pACYC184-rnpA(sRNA) at an OD600of 0.4.)

FIG. 4A shows the results of analyzing expression levels of a silkprotein consisting of 96 repeats by SDS-PAGE. (lane 1: a marker showingprotein standard molecular weight; lane 2: the result of inducingprotein expression in a strain transformed with pSH96 at an OD₆₀₀ of0.4; and lane 3: the result of inducing protein expression in a straintransformed with the plasmid pSH96 and pACYC184-rnpA(sRNA) at an OD₆₀₀of 0.4.)

FIG. 4B shows the results obtained by inducing protein expression instrains transformed with the plasmid pSH96 and the plasmid pSH96 pluspTetgly2glyAN-rnpA(sRNA), respectively, at an OD₆₀₀ of 0.4, analyzingprotein expression levels by SDS-PAGE, quantifying the proteinexpression levels using a densitometer, and then averaging the proteinexpression levels.

FIG. 5 shows the result of analyzing expression levels of a silk proteinconsisting of 128 repeats by SDS-PAGE. (lane 1: a marker showing proteinstandard molecular weight; lane 2: the result of inducing proteinexpression in a strain transformed with pSH128 at an OD600 of 0.4; andlane 3: the result of inducing protein expression in a straintransformed with pSH128 and pACYC184-rnpA(sRNA) at an OD₆₀₀ of 0.4.)

FIG. 6 is a graph showing the results of examining whether the level ofintracellular mRNA is increased by reducing expression of the rnpA gene.

FIG. 7A is a graph showing the amount of a silk protein consisting of 96repeats, produced by fed-batch culture, and FIG. 7B shows the results ofelectrophoresis of the protein.

FIG. 8 shows the results of electrophoresis performed using a system forreducing expression of the rnpA gene according to the present inventionin order to confirm increased expressions of difficult-to-expressproteins other than the silk protein. Specifically, electrophoresisimage (a) shows the results of analyzing the expression level of malicenzyme (SfcA) by SDS-PAGE. (lane 1: a marker showing protein standardmolecular weight; lane 2: the result of inducing protein expression innon-transformed BL21(DE3); lanes 3 and 4: the results of inducingprotein expression in a strain transformed with SfcA at an OD600 of 0.4;and lanes 5 and 6: the results of inducing protein expression in astrain transformed with SfcA and rnpA(sRNA) at an OD₆₀₀ of 0.4).Electrophoresis image (b) shows the results of analyzing the expressionlevel of Cat2 by SDS-PAGE. (lane 1: a marker showing protein standardmolecular weight; lanes 2 and 3: the results of inducing proteinexpression in a strain transformed with Cat2 at an OD₆₀₀ of 0.4; andlanes 4 and 5: the results of inducing protein expression in a straintransformed with Cat2-rnpA(sRNA) at an OD₆₀₀ of 0.4). Electrophoresisimage (c) shows the results of analyzing the expression level of SrtA bySDS-PAGE. (lane 1: a marker showing protein standard molecular weight;lanes 2 and 3: the results of inducing protein expression in a straintransformed with SrtA at an OD₆₀₀ of 0.4; and lanes 4 and 5: the resultsof inducing protein expression in a strain transformed withsrtA-rnpA(sRNA) at an OD600 of 0.4). Electrophoresis image (d) shows theresults of analyzing the expression level of CYP73A5 by SDS-PAGE. (lane1: a marker showing protein standard molecular weight; lanes 2 and 3:the results of inducing protein expression in a strain transformed withCYP73A5at an OD600 of 0.4; and lanes 4 and 5: the results of inducingprotein expression in a strain transformed with CYP73A5-rnpA(sRNA) at anOD600 of 0.4). Electrophoresis image (e) shows the results of analyzingthe expression level of CYP98A3 by SDS-PAGE. (lane 1: a marker showingprotein standard molecular weight; lanes 2 and 3: the results ofinducing protein expression in a strain transformed with CYP98A3 at anOD₆₀₀ of 0.4; and lanes 4 and 5: the results of inducing proteinexpression in a strain transformed with CYP98A3-rnpA(sRNA) at an OD₆₀₀of 0.4).

BEST MODE FOR CARRYING OUT THE INVENTION

The present inventors have developed a method for improving expressionof a difficult-to-express recombinant protein, which was not easilyproduced in the prior art, in a recombinant microorganism by reducingexpression of ribonuclease P to increase the mRNA level of a usefulprotein in cells.

In the present invention, expression of the rnpA gene that is acomponent of ribonuclease P was reduced using a sRNA system comprisingsRNA, and as a result, it was shown that expression of ahigh-molecular-weight silk protein that is a difficult-to-expressprotein was increased dramatically.

As used herein, the term “difficult-to-express protein” refers to aprotein having a molecular weight of 50 kDa or more.

Therefore, in one aspect, the present invention is directed to arecombinant vector for expressing a target protein, which comprises agene encoding the target protein and an sRNA against a gene encodingribonuclease P, and to a recombinant microorganism transformed with therecombinant vector.

In the present invention, the target protein may be a protein selectedfrom among difficult-to-express proteins silk proteins, antibodies,enzymes, cytochromes, and sortase A, but is not limited thereto.

In the present invention, the sRNA may be an sRNA against an rnpA gene,and may have a nucleotide sequence set forth in any one of SEQ ID NOs: 1to 3, but is not limited thereto as long as it reduces the expression ofan rnpA gene.

In the present invention, examples of microorganisms for production ofproteins that can be used may include Escherichia, Pseudomonas,Saccharomyces, and the like, and may be preferably an Escherichiamicroorganism, most preferably E. coli. In particular, it isadvantageous that E. coli can be easily industrialized since geneticinformation and culture conditions that are widely used industrially arevery well known.

In an example of the present invention, a recombinant E. coli strain wasconstructed by transformation with a gene encoding a silk proteinresulting from modification of a dragline silk protein obtained fromNephila clavipes, a nucleotide sequence encoding glycine tRNA, and ansRNA for reducing expression of the RnpA that is a component ofribonuclease P. The constructed recombinant E. coli strain was cultured.As a result, it could be seen that the protein of the silk proteinincreased 3-fold or more. In addition, it was shown that expression of asilk protein consisting of 16, 32, 48, 64, 80, 96, 112 and 128 repeatsof a specific amino acid sequence (SGRGGLGGTGAGMAAAAAMGGAGQGGYGGLGSQG)(SEQ ID NO: 19) was dramatically increased by reducing expression of thernpA gene that is a component of ribonuclease P. Furthermore, it wasshown that expressions of eGFP, SfcA and a full-length IgG antibody werealso dramatically increased by reducing expression of the rnpA gene thatis a component of ribonuclease P.

In view of the results indicating that the production of a longrecombinant protein or a difficult-to-express protein is increased byreducing expression of the rnpA gene that is a component of ribonucleaseP, it will be obvious to those skilled in the art that the degradationof messenger RNA (mRNA) has a great influence on protein expression andthat overexpression of a target protein can be effectively achieved byreducing expression of a gene associated with messenger RNA (mRNA)degradation.

In another example of the present invention, the expressions of a malicenzyme-encoding gene (sfcA), a sortase A-encoding gene (srtA), a4-hydroxybutyrate coenzyme A transferase-encoding gene (Cat2) and thecytochrome P450 gene (CYP73A5; cinnamate 4-hydrosylase and CYP98A3) wereexamined using the system for reducing expression of the rnpA geneaccording to the present invention. As a result, it was shown that astrain expressing an sRNA against the rnpA gene together with the geneencoding each of the proteins showed a higher protein expression levelcompared to a strain expressing no sRNA (FIG. 8; see electrophoresisimages (a) through (e)).

In another aspect, the present invention is directed to a recombinantmicroorganism into which a gene encoding a target protein and an sRNAagainst a gene encoding ribonuclease P are introduced.

In the present invention, the gene encoding the target protein and thesRNA against the gene encoding ribonuclease P may be present in therespective vectors or may be incorporated into the microbial chromosome.

In still another aspect, the present invention is directed to a methodfor producing a difficult-to-express target protein, the methodcomprising the steps of: (a) producing the target protein by culturingthe recombinant microorganism and inducing expression of the targetprotein in the recombinant microorganism; and (b) recovering theproduced target protein.

In an example of the present invention, a recombinant E. coli strain wasconstructed by transformation with a gene encoding a silk protein, anucleotide sequence encoding glycine tRNA, and an sRNA for reducingexpression of the rnpA gene that is a component of ribonuclease P. Theconstructed recombinant E. coli strain was cultured. As a result, itcould be seen that the protein of the silk protein increased 3-fold ormore.

In another example of the present invention, it was shown that, when agene encoding eGFP protein, a gene encoding sfcA protein and a geneencoding a full-length IgG antibody protein were co-expressed with ansRNA against the rnpA gene, expressions of the proteins increased.

In the present invention, preferably, the target protein may be a largeprotein having a molecular weight of 50 kDa or more as adifficult-to-express protein. For example, the target protein may be aprotein selected from a group consisting of silk proteins, antibodies,enzymes, cytochromes, and sortase A, but is not limited thereto.

In still another aspect, the present invention is directed to a methodfor producing a target protein, comprising the steps of: expressing andproducing the target protein by culturing a recombinant microorganisminto which a gene encoding the target protein is introduced; andrecovering the produced target protein, wherein expression ofribonuclease P in the recombinant microorganism is reduced to increaseexpression of the target protein.

In the present invention, the target protein may be adifficult-to-express protein or a protein having a molecular weight of50 kDa or more, and a substance of inhibiting the expression ofribonuclease P may be added.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Generally, the nomenclatureused herein and the experiment methods, which will be described below,are those well known and commonly employed in the art.

The definition of main terms used in the detailed description of theinvention is as follows.

As used herein, the term “sRNA (small RNA)” refers to a short-lengthRNA, which is usually 200 or less nucleotides in length, which is nottranslated into protein and effectively inhibits the translation of aspecific mRNA by complementary binding.

As used herein, the term “ribosome binding site” refers to a site whereribosome binds to mRNA for the transcription of the mRNA.

As used herein, the term “gene” is intended to have the broadestmeaning, and the gene can encode a structural protein or a regulatoryprotein. Herein, the regulatory protein includes a transcriptionalfactor, a heat shock protein, or a protein that is involved in DNA/RNAreplication, transcription and/or translation. Also, the target genewhose expression is to be inhibited may be present as anextrachromosomal element.

As used herein, the term “vector” means a DNA construct containing a DNAsequence operably linked to a suitable control sequence capable ofeffecting the expression of the DNA in a suitable host. The vector maybe a plasmid, a phage particle, or simply a potential genomic insert.Once incorporated into a suitable host, the vector may replicate andfunction independently of the host genome, or may in some instances,integrate into the genome itself. In the present specification,“plasmid” and “vector” are sometimes used interchangeably, as theplasmid is the most commonly used form of vector. For the purpose of thepresent invention, the plasmid vector is preferably used. A typicalplasmid vector which can be used for this purpose contains: (a) areplication origin by which replication occurs efficiently such thatseveral hundred plasmid vectors per host cell are created; (b) anantibiotic-resistant gene by which host cells transformed with theplasmid vector can be selected; and (c) restriction enzyme cutting sitesinto which foreign DNA fragments can be inserted. Even if suitablerestriction enzyme cutting sites are not present in the vector, the useof a conventional synthetic oligonucleotide adaptor or linker enablesthe easy ligation between the vector and the foreign DNA fragments.After ligation, the vector should be transformed into suitable hostcells. The transformation can be easily achieved by the calcium chloridemethod or electroporation (Neumann, et al., EMBO J., 1:841, 1982). Apublicly known expression vector in the art may be used as a vector forexpressing sRNA according to the present invention.

A nucleic acid sequence is operably linked when it is placed intoarranged in a functional relationship with another nucleic acidsequence. The nucleotide sequence may be a gene and a controlsequence(s) linked to be capable of expressing the gene when a suitablemolecule binds to a control sequence(s) (e.g., transcription-activatingprotein). For example, DNA for a pre-sequence or a secretory leader isoperably linked to a DNA encoding a polypeptide when expressed as apre-protein participating in secretion of the polypeptide; a promoter oran enhancer is operably linked to a coding sequence when affecting thetranscription of the sequence; or a ribosome binding site is operablylinked to a coding sequence when affecting the transcription of thesequence, or to a coding sequence when arranged to facilitatetranslation. Generally, the term “operably linked” means that the DNAlinked sequences are contiguous, and in the case of the secretoryleader, are contiguous and present in a reading frame. However, anenhancer is not necessarily contiguous. The linkage between thesesequences is performed by ligation at a convenient restriction enzymesite. However, when this site does not exist, a syntheticoligonucleotide adaptor or a linker is used according to a conventionalmethod.

In addition, the present invention is directed to a recombinantmicroorganism into which an expression vector comprising a nucleic acidencoding the sRNA is introduced. As used herein, the term“transformation” means that DNA can be replicated as a factor outside ofchromosome or by means of completion of the entire chromosome byintroducing DNA as a host.

Of course, it should be understood that all vectors and expressioncontrol sequences do not equally function to express DNA sequencesaccording to the present invention. Similarly, all hosts do not equallyfunction with respect to the same expression system. However, oneskilled in the art may appropriately select from a group consisting ofvarious vectors, expression control sequences, and hosts without eitherdeparting from the scope of the present invention or bearing excessiveexperimental burden. For example, a vector must be selected consideringa host, because the vector must be replicated in the host. Specifically,the copy number of the vector, the ability of regulating the copy numberand the expression of other protein encoded by the corresponding vector(e.g., the expression of an antibiotic marker) should also beconsidered.

EXAMPLES

Hereinafter, the present invention will be described in further detailwith reference to examples. It will be obvious to a person havingordinary skill in the art that these examples are for illustrativepurposes only and are not to be construed to limit the scope of thepresent invention.

Example 1: Construction of Recombinant Plasmid pTetgly2glyAN-rnpA(sRNA)

All procedures for gene manipulation followed standardized methods(Sambrook et al., Molecular cloning: a laboratory manual, 2nd Ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

To introduce an sRNA system into a pTetlgy2glyAN vector (Korean PatentNo. 1147860), genetic manipulation was performed, and to obtain aribosome binding site that is involved in translation of the rnpA gene,inverse-PCR was performed using primers of SEQ ID NOs: 4 and 5, therebyobtaining an rnpA sRNA (SEQ ID NO: 1).

SEQ ID NO: 4: 5′-cctgggaaatgcgagcttaaccactttctgttgggccattgcattg- 3′SEQ ID NO: 5: 5′-GCAACCATTATCACCGCCA-3

The PCR reaction was performed using Pfu polymerase (SolGent, Korea)under the following conditions: pre-denaturation at 95° C. for 5 min,and then 28 cycles, each consisting of denaturation at 95° C. for 30sec, annealing at 57° C. for 180 sec, and extension at 72° C. for 60sec, followed by final extension at 72° C. for 5 min.

The PCR product was electrophoresed on agarose gel to obtain a purified5000-bp PCR product. The purified PCR product was incubated with therestriction enzyme DpnI (New England Biolabs, USA) for 1 hour, and thenligated with a pTetlgy2glyAN by T4 DNA ligase(Roche, Germany), and theresulting vector was transformed into E. coli dH5α (FhuA2 lac(del)U169phoA glnV44 Φ80′ lacZ(del)M15 gyrA96 recAl relAl endAl thi-1 hsdR17,Invitrogen).

The transformed strain was selected on an LB agar solid medium (tryptone10 g/L, yeast extract 5 g/L, NaCl 5 g/L, and agar 15 g/L) containing 34mg/L of chloramphenicol, thereby obtaining the recombinant plasmidpTetgly2glyAN-rnpA(sRNA) (FIG. 1). The constructed recombinant plasmidwas confirmed by cleaving with restriction enzymes and DNA sequencing.

Example 2: Construction of Recombinant Plasmids Containing the GeneEncoding High-Molecular-Weight Silk Protein

In order to construct a recombinant plasmid containing 32 repeats of agene that encodes a silk protein resulting from modification of aNephila clavipes derived dragline silk protein that is adifficult-to-express protein having a large size, the plasmid pSH16a(Lee et al., Theories and Applications of Chem. Eng., 8:3969, 2002)consisting of 16 repeats of the gene encoding the silk protein wasdigested with the restriction enzyme SpeI and NheI to obtain a 1.7-kbfragment. The fragment was ligated with the plasmid pSH16a digested withthe restriction enzyme SpeI, thereby obtaining the recombinant plasmidpSH32. The direction of the ligated insert was determined by digestionwith the restriction enzyme SpeI and NheI (New England Biolabs, USA).

Similarly, the plasmid pSH16a was digested with the restriction enzymeSpeI and NheI to obtain a 1.7-kb fragment which was then ligated withthe plasmid pSH32 digested with the restriction enzyme SpeI, therebyconstructing the recombinant plasmid pSH48. The recombinant plasmidpSH32 was digested with the restriction enzyme SpeI and NheI to obtain a3.4-kb fragment which was then ligated with the plasmid pSH64 digestedwith the restriction enzyme SpeI, thereby constructing the recombinantplasmid pSH64. The plasmid pSH48 was digested with the restrictionenzyme SpeI and NheI to obtain a 5.1-kb fragment which was then ligatedwith the plasmid pSH32 digested with the restriction enzyme SpeI,thereby constructing the recombinant plasmid pSH80. The plasmid pSH16was digested with the restriction enzyme SpeI and NheI to obtain a1.74-kb fragment which was then ligated with the plasmid pSH80 digestedwith the restriction enzyme SpeI, thereby constructing the recombinantplasmid pSH96. The plasmid pSH32 was digested with the restrictionenzyme SpeI and NheI to obtain a 3.4-kb fragment which was then ligatedwith the plasmid pSH80 digested with the restriction enzyme SpeI,thereby constructing the recombinant plasmid pSH112. The plasmid pSH64was digested with the restriction enzyme SpeI and NheI to obtain a7.8-kb fragment which was then ligated with the plasmid pSH64 digestedwith the restriction enzyme SpeI, thereby constructing the recombinantplasmid pSH128. The direction of each ligated insert was determined bydigestion with the restriction enzyme SpeI and NheI.

Example 3: Examination of the Increase in Silk Protein Expression Causedby the Reduction in Expression of rnpA Gene by sRNA System

In order to examine the effect of co-overexpressing the glycine tRNAgene and reducing the expression of the ribonuclease P (rnpA) gene byuse of the sRNA system in silk protein production, E. coli BL21 (DE3)(F-ompT hsdSB(rB- mB-) gal dcm (DE3) a prophage carrying the T7 RNApolymerase gene) (New England Biolabs, USA) was transformed with theplasmid pTetgly2glyAN-rnpA(sRNA) obtained in Example 1 and each of theplasmid pSH16, pSH32, pSH48, pSH64, pSH80, pSH96 and pSH112 containing16, 32, 48, 64, 80, 96 and 112 repeats of the silk protein-encodinggene, respectively.

As a control, E. coli BL21 (DE3), transformed with the plasmid pACYC184and each of pSH16, pSH32, pSH64, pSH96 and pSH128, was used. Each of thetransformed strains was seeded into 10 ml of an LB liquid medium(tryptone 10 g/L, yeast extract 5 g/L, and NaCl 5 g/L)(containing 34mg/L of chloramphenicol, 25 mg/L of kanamycin and 1% arabinose) andshake-cultured at 25° C. and 220 rpm. Next, each of the strains wasshake-cultured under the above-described medium conditions at 37° C. and220 rpm. When the culture reached an OD₆₀₀ of 0.4, 1 mM IPTG was addedto the medium to induce expression of the silk protein gene. At 4 hoursafter induction of the expression, the culture was sampled andcentrifuged at 4° C. and 10,000 g for 10 minutes, and the obtained cellpellets were dissolved in TE buffer and 5× Laemmli sample buffer. Thesame amount (0.024 mg) of the sample was separated using 10% SDS-PAGEgel, after which it was stained with Coomassie brilliant blue R250(Bio-Rad, USA) solution and quantified using the GS-710 CalibratedImaging Densitometer (Bio-Rad, USA) (see FIGS. 2, 3, 4A, and 4B).

Furthermore, expression of the strain transformed with the plasmid pSH96and pTetgly2glyAN-rnpA(sRNA) was induced at an OD₆₀₀ 0.4, after whichprotein expression was analyzed by SDS-PAGE, quantified using adensitometer and averaged. The results are shown in FIG. 4B. Inaddition, the results of expression performed using the plasmid pSH128are shown in FIG. 5.

As a result, it could be seen that, due to the reduction in expressionof ribonuclease P (RnpA), expression of the silk protein consisting of16 repeats was increased by about 150% compared to the control, andexpression of the silk protein consisting of 32 repeats was increased byabout 300% compared to the control. Furthermore, expression of the silkprotein consisting of 48 repeats was increased by about 300%, andexpression of the silk protein consisting of 64 repeats was increased byabout 300%. In addition, expression of the silk protein consisting of 80repeats was increased by about 300%, expression of the silk proteinconsisting of 112 repeats was increased by about 200%, and expression ofthe silk protein consisting of 128 repeats was increased by about 150%.

Example 4: Examination of the Increase in mRNA Level Caused by theReduction in Expression of rnpA Gene by sRNA System

In this Example, in order to prevent the degradation of messenger RNA inthe production of recombinant, difficult-to-express and useful proteins,expression of the rnpA gene that is a component of ribonuclease P wasreduced by introduction of the sRNA system. In addition, whether thelevel of intracellular mRNA would be increased by the reduction inexpression of the rnpA gene was examined.

Specifically, E. coli BL21 (DE3) (F-ompT hsdSB(rB- mB-) gal dcm (DE3) aprophage carrying the T7 RNA polymerase gene) (New England Biolabs, USA)was transformed with the plasmid pTetgly2glyAN-rnpA(sRNA) obtained inExample 1 and pSH32.

As a control, E. coli BL21 (DE3), transformed with the plasmid pACYC184and pSH32, was used. Each of the transformed strains was seeded into 10ml of an LB liquid medium (tryptone 10 g/L, yeast extract 5 g/L, andNaCl 5 g/L) (containing 34 mg/L of chloramphenicol, 25 mg/L of kanamycinand 1% arabinose) and shake-cultured at 25° C. and 220 rpm. Next, eachof the strains was shake-cultured under the above-described mediumconditions at 37° C. and 220 rpm. When the culture reached an OD₆₀₀ of0.4, 1 mM IPTG was added to the medium to induce expression of the silkprotein gene. At 4 hours after induction of the expression, the culturewas sampled and centrifuged at 4° C. and 10,000 g for 10 minutes toobtain cell pellets, and RNA was extracted from the cell pellets.

For RNA extraction, 1 ml of Trizol was added to the cell pellet, afterwhich the cell pellet solution was stirred for 1 minute and allowed tostand at room temperature for 5 minutes. Next, chloroform in an amountequal to 20% of the total volume was added to the cell pellet solutionwhich was then stirred for 15 seconds and centrifuged at 13,000 rpm for15 minutes. The produced supernatant was transferred into a fresh tube,and the same amount of isopropanol was added thereto and carefullystirred. Then, the mixture was centrifuged at 13,000 rpm for 30 minutes,and the supernatant was discarded. Next, 1 ml of 70% ethanol was addedto the remaining material which was then centrifuged at 13,000 rpm for 5minutes, and the above procedure was repeated again. Next, the resultingmaterial was dried at room temperature and dissolved in 30 μl ofRNase-free distilled water. In order to replace the extracted RNA withcomplementary DNA (cDNA), qPCR was performed using Rocketstrip (Bioneer,Korea). The extracted RNA was adjusted to 500 ng, and 2 μl of dN9 primerwas added thereto. Then, the reaction mixture was adjusted to a totalvolume of 20 μl using RNase-free distilled water. The PCR reaction wasperformed under the following conditions: initial annealing at 30° C.for 150 seconds, cDNA synthesis at 60° C. for 1 hour, and then heatinactivation at 95° C. for 300 seconds.

To perform RT-PCR using the prepared cDNA, a reaction mixture having atotal volume of 20 μl contained 8 μl of RNase-free distilled water, 10μl of SYBR Green Mastermix (Enzynomics, Korea), 1 μl of a 1/10 dilutionof the cDNA, and 0.5 μl of each primer. The RT-PCR reaction wasperformed under the following conditions: initial activation at 95° C.for 10 minutes, and then 45 cycles, each consisting of denaturation at95° C. for 30 sec, annealing at 60° C. for 30 sec, and extension at 72°C. for 30 sec. To determine the melting curve, the temperature wasincreased from 55° C. to 95° C. by 0.5° C., and the reaction mixture wasmaintained at each temperature for 5 seconds.

As a result, as shown in FIG. 6, in the case of cells in whichexpression of ribonuclease P (RnpA) was reduced, the level of mRNA was24-fold higher than that in the control. This suggests that thedegradation of mRNA is prevented by reducing expression of ribonucleaseP (RnpA).

Example 5: Examination of Increase in Spider Silk Protein Production byFed-Batch Fermentation

The strain transformed with the plasmid pSH96 and pACYC184-rnpA(sRNA),constructed in Example 3, was cultured by fed-batch fermentation, and anincrease in protein production in the strain was examined.

For fed-batch fermentation, a 6.6-L fermentor (Bioflo 3000; NewBrunswick Scientific Co.) was used, and 1.6 L of R/2 medium, 10 g/L ofglucose, 0.7 g/L of MgSO₄.7H₂O, and an antibiotic (50 μg/mL kanamycinand/or 35 μg/mL chloramphenicol) were added to the strain in thefermentor. To adjust the dissolved oxygen level to 40%, air saturationwas adjusted while the agitation speed was increased to 1000 rpm. Afeeding solution was composed of 700 g/L of glucose and 20 g/LMgSO₄.7H₂O, and the pH of the culture was adjusted using 28% (v/v)ammonia solution. When the OD600 reached about 70, 1 mM IPTG was addedto the culture to induce protein expression. For 8 hours after inductionof the expression, the culture was sampled at 2-hour intervals. Cellproliferation and the concentration of the protein obtained are shown inFIG. 7. As can be seen in FIG. 7, the protein could be obtained at aconcentration of 0.9 g/L, which was 30% higher than the previously knownconcentration (0.7 g/L).

Example 6: Examination of the Increase in Expression of Other Proteinsby Reduction in Expression of rnpA Gene

Using the system for reducing rnpA gene expression according to thepresent invention, expressions of the gene (sfcA, SEQ ID NO: 6) encodingmalic enzyme, the gene (srtA, SEQ ID NO: 7) encoding sortase A, the gene(cat2, SEQ ID NO: 8) encoding 4-hydroxybutyrate coenzyme A transferaseand the cytochrome P450 gene (CYP73A5; cinnamate 4-hydrosylase:SEQ IDNO: 9 and CYP98A3:SEQ ID NO: 10) were analyzed, in addition toexpression of the gene encoding silk protein. The Sortase A gene wassynthesized by Bioneer (Korea), and the sfcA, cat2, CYP73A5 and CYP98A3genes were obtained by PCR using the following primers.

sfcA_F: (SEQ ID NO: 11) 5′-CCATGGATATTCAAAAAAGAGTGAGT-3′ sfcA_R:(SEQ ID NO: 12) 5′-TCTAGATTAGATGGAGGTACGGCGGTA-3′ Cat2_F:(SEQ ID NO: 13) 5′-GAATTCATGGAGTGGGAAGAGATATATA-3′ Cat2_R:(SEQ ID NO: 14) 5′-GGTACCCTAAAATCTCTTTTTAAATTCATT-3′ CYP73A5_F:(SEQ ID NO: 15) 5′-GCGAAGCTTACAGTTCCTTGGTTTCATAA-3′ CYP73A5_R:(SEQ ID NO: 16) 5′-GTACATATGATGGACCTCCTCTTGCTGG-3′ CYP98A3_F:(SEQ ID NO: 17) 5′-GGATCCATGTCGTGGTTTCTAATAGC-3′ CYP98A3_R:(SEQ ID NO: 18) 5′-GAATTCTTACATATCGTAAGGCACGC-3′

RnpA sRNA (SEQ ID NO: 1) and each of the sfcA, srtA and cat2 genes wereinserted into pET30a (+)(Addgene, USA) and pTac15k (Addgene, USA)respectively to obtain recombinant vectors. Each of the vectors wastransformed into an E. coli BL21 (DE3) strain. As a control, a straintransformed with a vector obtained by inserting each of the sfcA, srtAand cat2 genes into pET30a and pTac15k was used.

Each of the transformed strains was seeded into 10 ml of an LB liquidmedium (tryptone 10 g/L, yeast extract 5 g/L, and NaCl 5 g/L)(containing 34 mg/L of chloramphenicol, 25 mg/L of kanamycin and 1%arabinose) and shake-cultured at 25° C. and 220 rpm. Next, each of thestrains was shake-cultured under the above-described medium conditionsat 37° C. and 220 rpm. When the culture reached an OD₆₀₀ of 0.4, 1 mMIPTG was added to the medium to induce expression of each protein. At 4hours after induction of the expression, the culture was sampled andcentrifuged at 4° C. and 10,000 g for 10 minutes to obtain cell pellets.The cell pellets were dissolved in TE buffer and 5× Laemmli samplebuffer. The same amount (0.024 mg) of the sample was separated on 10%SDS-PAGE gel and stained with Coomassie brilliant blue R250 (Bio-Rad,USA) solution.

As a result, as can be seen in FIG. 8 (see electrophoresis images (a)through (e) therein), the expression levels of malic enzyme, sortase A,4-hydroxybutyrate coenzyme A transferase (Cat2) and cytochrome P450(cinnamate 4-hydrosylase and CYP98A3), expressed together with the RnpAsRNA, were significantly higher than the expression levels of theproteins expressed in the absence of the RnpA sRNA.

Although the present invention has been described in detail withreference to the specific features, it will be apparent to those skilledin the art that this description is only for a preferred embodiment anddoes not limit the scope of the present invention. Thus, the substantialscope of the present invention will be defined by the appended claimsand equivalents thereof.

INDUSTRIAL APPLICABILITY

According to the present invention, expression of a target protein,particularly a difficult-to-express protein having a high molecularweight can be dramatically increased by reducing expression of the rnpAgene so that the present invention is useful for increasing theproductivity of proteins.

What is claimed is:
 1. A method for producing a target protein,comprising the steps of: producing the target protein by culturing arecombinant microorganism into which a gene encoding the target proteinis introduced; and recovering the produced target protein, wherein asubstance of reducing expression of ribonuclease P is added in theculture to increase expression of the target protein.
 2. The method ofclaim 1, wherein the target protein is a difficult-to-express protein ora protein having a molecular weight of 50 kDa or more.
 3. The method ofclaim 1, wherein the target protein is a silk protein, antibody,cytochrome, enzyme, or sortase A.
 4. The method of claim 1, wherein saidsubstance is an sRNA.
 5. The method of claim 1, wherein said sRNA is ansRNA against an rnpA gene.
 6. The method of claim 1, wherein said sRNAhas a nucleotide sequence of any one of SEQ ID NOS: 1 to 3.